NOVEL USE OF PROLIFERATING CELL NUCLEAR ANTIGEN AS A BIOMARKER OF METASTATIC CANCER

Novel biomarkers to identify metastatic tumor cells are needed to better identify these cells and to appropriately choose therapeutic measures. Cancer stem cells are believed to be responsible for metastasis and relapse after therapy. We have identified novel expression of Proliferating Cell Nuclear Antigen on the cell surface of tumors and characterized these cells as potential cancer stem cells. This research may facilitate the generation of novel immune based cancer therapies to specifically identify and target metastatic tumor cells. Purpose (a): Primary tumors account for 10% of cancer related deaths. Thus, identifying novel biomarkers on tumor cells which resist treatment or potentially become metastatic is vital. Proliferating Cell Nuclear Antigen (PCNA) has traditionally been used as a biomarker to identify and grade tumor biopsies based on PCNA's involvement in DNA replication. Typically located intracellularly, we have recently identified PCNA at the cell surface. When recognized by the Natural Killer (NK) cell receptor, NKp44, PCNA inhibits NK cell effector functions, allowing tumor cells to escape immunosurveillance. We have characterized tumor cells expressing cell surface PCNA to evaluate the use of cell surface PCNA as a potential marker of cancer stem cells, believed to be responsible for relapse and metastasis. Methods (b): We analyzed extracellular PCNA expressing tumor cells for expression of vimentin by confocal microscopy and expression of CD44 and CD24 by flow cytometry, which mark cancer stem cells. We also analyzed these cells for expression of genes which can induce formation of cancer stem cells or maintain stem cell characteristics by real time PCR. Finally, since stem cells are often quiescent, we analyzed cell cycle progression of these cells using propidium iodide and flow cytometry analysis. Results (c): Expression of vimentin is exclusive to cells expressing extracellular PCNA. These cells also express intermediate levels of CD44, which marks metastatic cells in vivo, and differentially express genetic markers of cancer stem cells. Populations of tumor cells expressing PCNA at the cell surface were enriched for cells in the G2/M phase of the cell cycle. Conclusions (d): Extracellular PCNA may be a marker for metastatic cancer stem cells based on intermediate expression of CD44, concomitant expression of vimentin, and expression of genetic markers. Alternatively, extracellular PCNA marks cells in the G2/M phase. Further studies in mouse models will be needed to confirm extracellular PCNA as a marker for cancer stem cells.